In situ hybridization assays have become increasingly important diagnostic tools in evaluating a variety of pathologic conditions. The technique combines the classical efficacy of morphologic visualization of a selected cell population with the ability to simultaneously detect the presence and/or expression of specific genes.
An early paper, Lawrence and Singer, Nuc. Acids Res., 13: 1777 (1985) describes the use of plasmid probes, isotopically labelled, in detecting action gene expression. This paper also describes experiments for optimizing the pretreatment conditions in preparing cells for in situ hybridization. It was found that pretreatment with 5% proteinase K followed by immersion in 2% paraformaldehyde permitted satisfactory hybridization without undue distortion of cellular morphology. A nonisotopic probe detection system for action gene expression was reported by Singer and Ward, PNAS, 79: 7331 (1982).
The sensitivity of in situ hybridization was evaluated by Hafen, et al., EMBO Journal, 2: 617 (1983). Utilizing a tritiated probe, it was possible to detect about 100 complementary RNA molecules per cell after three days of autoradiographic exposure. Brigati et al., Virology, 126: 32 (1983) demonstrated the feasibility of using biotinylated probes in an in situ hybridization assay of paraffin block specimens coupled to immunohistochemical detection mediated by horseradish peroxidase. For a detailed review of the applications of in situ hybridization, see In Situ Hybridization, Principles and Practice, eds. Polak & McGee, Oxford University Press, 1990.
Of particular interest is the use of in situ hybridization techniques in the investigation of human papilloma virus (HPV) infections, and the role of the virus in transformation of cervical cells. Herrington, et al. demonstrated that in situ hybridization was capable of discriminating among closely homologous HPV types 6 and 11. However, these investigators found that hybridization conditions in solution and in situ were very different, and kinetic equations derived from solution data were not predictive of the hybridization properties of selected probes in situ.
The inherent limitation in this and other assay techniques based on direct hybridization of native RNA or DNA with labelled probes is copy number. If the number of target molecules present in cells is very low, on the order of less than 100 to 1000, then the risk of a false negative test is very great. In order to overcome this limitation, Haase et al., PNAS, 87: 4971 (1990) recently employed the technique of polymerase chain reaction (PCR) to amplify the low copy number of certain lentiviruses present in cells, so that sufficient target is available for hybridization and subsequent visualization. While this technique was shown to overcome the problem of low copy number, it is also evident that significant natural morophology is destroyed because of the harsh temperature shifts between 65.degree. C. and 95.degree. C. involved in PCR cycling.